Specific inhibition of fMAPK pathway output by <i>MPT5</i>.

<p>(A) Diploid strains with either a minimal filamentation-MAPK-pathway output reporter (FRE-GFP) or a mating-MAPK reporter (PRE-GFP) and the indicated genotypes were grown under yeast-form conditions and subjected to flow cytofluorometry. (B) The morphology and fluorescence of <i>MPT5</i>+ and <i>mpt5</i>Δ diploid cells with FRE-GFP were imaged by transmitted light and confocal fluorescence microscopy.</p

UTR sequences and repression by <i>MPT5</i>.

<p>Yeast strains were grown under yeast-form conditions. Protein and RNA extracts were prepared and analyzed by (A) western blot and (B) northern blot. Pgk protein and <i>U3</i> RNA served as loading controls.</p

<b>In vivo</b><i>TEC1</i> mRNA binding by Mpt5.

<p>An immunoprecipitate of epitope-tagged Mpt5 protein was subjected to reverse transcription and gene-specific polymerase chain reaction to detect the presence of bound mRNAs. Control experiments lacked either the epitope tag or reverse transcriptase.</p

Repression of yeast filamentous-form phenotypes by <i>MPT5</i>.

<p>(A–D) Diploid yeast were grown under yeast-form conditions (SCD liquid and SCD) or filamentous-form conditions (SLAD) and microscopically imaged. (E) Patches of yeast were grown on rich medium agar and subjected to a washing-off assay of adhesion.</p

Inhibition of Kss1-dependent phosophorylation of Ste7 by <i>MPT5</i>.

<p>Yeast strains were grown under yeast-form conditions. Protein extracts were analyzed by western blot, with Pgk serving as a loading control. The effect of <i>MPT5</i> deletion on Ste7 phosphorylation is independent of <i>RAS2</i> and <i>PHD1</i> (A) but depends on <i>KSS1</i> (B).</p

Top ranked motif for each of the eight expression clusters.

<p>Median fold change for each of the eight clusters is represented using blue lines and the values are shown on the left Y axes. Red lines represent Clover raw scores for the top ranked motif and the values are shown on the Y axes on the right. Based on a time-lagged correlation analysis (using optimum lag time for each motif), the correlation between the Clover score and the cluster-median expression levels are: UC1/V$CREBP1_Q2 - <i>R</i> = 0.828 (t = 2.0); UC2/V$IRF_Q6—<i>R</i> = 0.777 (t = 1.0004); UC3/V$IRF_Q6—<i>R</i> = 0.999 (t = 1.343); UC4/V$SP1_Q2_01—<i>R</i> = 0.8965 (t = 2.0); UC5/V$MYF_01—<i>R</i> = -0.900 (t = 1.001) DC1/V$NFY_01—<i>R</i> = 0.992 (t = 0.589); DC2/V$NFY_01—<i>R</i> = 0.916 (t = 2.0); DC3/V$ZFP281_01—<i>R</i> = 0.304 (t = 2.0).</p

Correlation between Clover scores and observed TF binding.

<p>Plots show relationship between Clover scores (y axes) and ChIP-seq counts (x axes) for motifs for IRF1 (V$IRF1), IRF8 (V$IRF8) and PU.1/SPI1 (V$PU1) for all eight clusters at 0, 2 and 4 h time points. Panes A-C show Clover scores and observed counts for enriched motifs (blue diamonds), correlation for those (black line) and Clover scores and counts for motifs that are not enriched (red squares). Panes D-F show Clover scores and ChIP-seq counts for the SPI1 motif separately for each cluster (three time points for each cluster).</p

Enrichment test results for ChIP-seq tags for specific TFs in HAc-valley regulatory elements within ±5 kbp of TSSs for genes in specific clusters.

<p>Enrichment test results for ChIP-seq tags for specific TFs in HAc-valley regulatory elements within ±5 kbp of TSSs for genes in specific clusters.</p

Identifying novel transcription factors involved in the inflammatory response by using binding site motif scanning in genomic regions defined by histone acetylation

<div><p>The innate immune response to pathogenic challenge is a complex, multi-staged process involving thousands of genes. While numerous transcription factors that act as master regulators of this response have been identified, the temporal complexity of gene expression changes in response to pathogen-associated molecular pattern receptor stimulation strongly suggest that additional layers of regulation remain to be uncovered. The evolved pathogen response program in mammalian innate immune cells is understood to reflect a compromise between the probability of clearing the infection and the extent of tissue damage and inflammatory sequelae it causes. Because of that, a key challenge to delineating the regulators that control the temporal inflammatory response is that an innate immune regulator that may confer a selective advantage in the wild may be dispensable in the lab setting. In order to better understand the complete transcriptional response of primary macrophages to the bacterial endotoxin lipopolysaccharide (LPS), we designed a method that integrates temporally resolved gene expression and chromatin-accessibility measurements from mouse macrophages. By correlating changes in transcription factor binding site motif enrichment scores, calculated within regions of accessible chromatin, with the average temporal expression profile of a gene cluster, we screened for transcriptional factors that regulate the cluster. We have validated our predictions of LPS-stimulated transcriptional regulators using ChIP-seq data for three transcription factors with experimentally confirmed functions in innate immunity. In addition, we predict a role in the macrophage LPS response for several novel transcription factors that have not previously been implicated in immune responses. This method is applicable to any experimental situation where temporal gene expression and chromatin-accessibility data are available.</p></div

AcH4 valleys and active promoter regions.

<p>(A) AcH4 valleys. ChIP-seq signal and smoothed ChIP-seq signal are shown by gray and black lines respectively. Green bars represent the locations of detected AcH4 valleys. (B) Active promoter regions, defined as regions where detected AcH4 valleys (short blue bars shown for different time point) overlap with the ±5,000bp region around TSS (long blue bar).</p